Method for treating restenosis with a2a adenosine receptor agonists

ABSTRACT

Agonists of A 2A  adenosine receptors in combination with rolipram, its derivatives or other Type IV phosphodiesterase (PDE) inhibitors are effective for the treatment of inflammatory diseases.

RELATED APPLICATION

[0001] This application is a continuation-in part of co-pending U.S.patent application Ser. No. 08/272,821, filed Jul. 22, 1994 to Linden etal., which is incorporated herein in its entirety by reference.

[0002] The present invention was made with the assistance of U.S.Government funding (NIH Grant R01-HL 37942). The U.S. Government mayhave some rights in this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to methods and compositions fortreating inflammatory diseases.

[0005] 2. Discussion of the Background

[0006] The release of inflammatory cytokines such as tumor necrosisfactor-alpha (TNFα) by leukocytes is a means by which the immune systemcombats pathogenic invasions, including infections. Cytokines stimulateneutrophils to enhance oxidative (e.g., superoxide and secondaryproducts) and nonoxidative (e.g., myeloperoxidase and other enzymes)inflammatory activity. Inappropriate and over-release of cytokines canproduce counterproductive exaggerated pathogenic effects through therelease of tissue damaging oxidative and nonoxidative products (Tracey,K. G., et al., J. Exp. Med., vol. 167, pp. 1211-1227 (1988); and Männel,D. N., et al., Rev. Infect. Dis., vol. 9 (suppl 5), pp. S602-S606(1987)).

[0007] For example, inflammatory cytokines have been shown to bepathogenic in: arthritis (Dinarello, C. A., Semin. Immunol., vol. 4, pp.133-45 (1992)); ischemia (Seekamp, A., et al., Agents-Actions-Supp.,vol. 41, pp. 137-52 (1993)); septic shock (Männel, D. N., et al., Rev.Infect. Dis., vol. 9, (suppl 5), pp. S602-S606 (1987)); asthma(Cembrzynska Nowak M., et al., Am. Rev. Respir. Dis., vol. 147, pp.291-5 (1993)); organ transplant rejection (Imagawa, D. K., et al.,Transplantation, vol. 51, pp. 57-62 (1991)); multiple sclerosis(Hartung, H. P., Ann. Neurol., vol. 33, pp. 591-6 (1993)); and AIDS(Matsuyama, T., et al., AIDS, vol. 5, pp. 1405-1417 (1991)). Inaddition, superoxide formation in leukocytes has been implicated inpromoting replication of the human immunodeficiency virus (HIV)(Legrand-Poels, S., et al., AIDS Res. Hum. Retroviruses, vol. 6, pp.1389-1397 (1990)).

[0008] It is well known that adenosine and some relatively nonspecificanalogs of adenosine decrease neutrophil production of inflammatoryoxidative products (Cronstein, B. N., et al., Ann. N.Y. Acad. Sci., vol.451, pp. 291-314 (1985); Roberts, P. A., et al., Biochem. J., vol. 227,pp. 66.9-674 (19-85); Schrier, D. J., et al., J. Immunol., vol. 137, pp.3284-3289 (1986); Cronstein, B. N., et al., Clinical Immunol. andImmunopath., vol. 42, pp. 76-85 (1987); Iannone, M. A., et al., inTopics and Perspectives in Adenosine Research, E. Gerlach et al., eds.,Springer-Verlag, Berlin, pp. 286-298 (1987); McGarrity, S. T., et al.,J. Leukocyte Biol., vol. 44, pp. 411421 (1988); De La Harpe, J., et al.,J. Immunol., vol. 143, pp. 596-602 (1989); McGarrity, S. T., et al., J.Immunol., vol. 142, pp. 1986-1994 (1989); and Nielson, C. P., et al.,Br. J.Pharmacol., vol. 97, pp. 882-888 (1989)). For example, adenosinehas been shown to inhibit superoxide release from neutrophils stimulatedby chemoattractants such as the synthetic mimic of bacterial peptides,f-met-leu-phe (fMLP), and the complement component C₅a (Cronstein, B.N.,et al., J. Immunol, vol. 135, pp. 1366-1371 (1985)). Adenosine candecrease the greatly enhanced oxidative burst of PMN (neutrophil) firstprimed with TNF-α (an inflammatory cytokine) and then stimulated by asecond stimulus such as f-met-leu-phe (Sullivan, G. W., et al., Clin.Res., vol. 41, p. 172A (1993)). There is evidence that in vivo adenosinehas anti-inflammatory activity (Firestein, G. S., et al., Clin. Res.,vol. 41, p. 170A (1993); and Cronstein, B. N., et al., Clin. Res., vol.41, p. 244A (1993)). Additionally, it has been reported that adenosinecan decrease the rate of HIV replication in a T-cell line (Sipka, S., etal., Acta. Biochim. Biopys. Hung., vol. 23, pp. 75-82 (1988)).

[0009] It has been suggested that there is more than one subtype ofadenosine receptor on neutrophils that have opposite effects onsuperoxide release (Cronstein, B. N., et al., J. Clin. Invest., vol. 85,pp. 1150-1157 (1990)). The existence of A_(2A) receptor on neutrophilswas originally demonstrated by Van Calker et al. (Van Calker, D., etal., Eur. J. Pharmacology, vol. 206, pp. 285-290 (1991)).

[0010] There has been progressive development of compounds that are moreand more potent and selective as agonists of A_(2A) adenosine receptorsbased on radioligand binding assays and physiological responses.Initially, compounds with little or no selectivity for A_(2A) receptorswere used, such as adenosine itself or 5′-carboxamides of adenosine,such as 5′-N-ethylcarboxamidoadenosine (NECA) (Cronstein, B. N., et al.,J. Immunol., vol. 135, pp. 1366-1371 (1985)). Later, it was shown thataddition of 2-alkylamino substituents increased potency and selectivity,e.g. CV1808 and CGS21680 (Jarvis, M. F., et al., J. Pharmacol. Exp.Ther., vol. 251, pp. 888-893 (1989)). 2-Alkoxy-substituted adenosinederivatives such as WRC-0090 are even more potent and selective asagonists on the coronary artery A_(2A) receptor (Ukena, M., et al., J.Med. Chem., vol. 34, pp. 1334-1339 (1991)). The 2-alkylhydrazinoadenosine derivatives, e.g. SHA 211 (also called WRC-0474) have alsobeen evaluated as agonists at the coronary artery A_(2A) receptor(Niiya, K., et al., J. Med. Chem., vol. 35, pp. 45574561(1992)).

[0011] There is one report of the combination of relatively nonspecificadenosine analogs, R-phenylisopropyladenosine (R-PIA) and2-chloroadenosine (Cl-Ado) with a phosphodiesterase (PDE) inhibitorresulting in a lowering of neutrophil oxidative activity (Iannone, M.A., et al., in Topics and Perspectives in Adenosine Research, E. Gerlachet al., Eds., Springer-Verlag, Berlin, pp. 286-298 (1987)). However,R-PIA and Cl-Ado analogs are actually more potent activators of A₁adenosine receptors than of A_(2A) adenosine receptors and, thus, arelikely to cause side effects due to activation of A₁ receptors oncardiac muscle and other tissues causing effects such as “heart block”.

[0012] Linden et al. Ser. No. 08/272,821 is based on the discovery thatinflammatory diseases may be effectively treated by the administrationof drugs which are selective agonists of A_(2A) adenosine receptors,preferably in combination with a phosphodiesterase inhibitor. Anembodiment of the Linden et al. invention provides a method for treatinginflammatory diseases by administering an effective amount of an A_(2A)adenosine receptor of the following formula:

[0013] wherein X is a group selected from the group consisting of —OR¹,—NR²R³, and —NH—N═R⁴;

[0014] wherein R¹ is C₁₋₄-alkyl; C₁₋₄-alkyl substituted with one or moreC₁₋₄-alkoxy groups, halogens (-fluorine, chlorine, or bromine), hydroxygroups, amino groups, mono(C₁₋₄-alkyl)amino groups, di(C₁₋₄,-alkyl)aminogroups, or C₆₋₁₀-aryl groups (wherein the aryl groups may be substitutedwith one or more halogens (fluorine, chlorine, or bromine), C₁₋₄-alkylgroups, hydroxy groups, amino groups, mono(C₁₋₄-alkyl)amino groups, ordi(C₁₋₄ alkyl)amino groups); C₆₋₁₀-aryl; or C₆₋₁₀-aryl substituted withone or more halogens (fluorine, chlorine, or bromine), hydroxy groups,amino groups, mono(C₁₋₄-alkyl)amino groups, or di(C₁₋₄ alkyl)aminogroups, or C₁₋₄-alkyl groups;

[0015] one of R² and R³ has the same meaning as R¹ and the other ishydrogen;

[0016] R⁴ is a group having the formula:

[0017] wherein each of R⁵ and R⁶ independently may be hydrogen,C₃₋₇-cycloalkyl, or any of the meanings of R¹, provided that R⁵ and R⁶are not both hydrogen; and

[0018] R is —CH₂OH, —CH₂H, —CO₂R⁷, or —C(═O)NR⁸R⁹; wherein R⁷ has thesame meaning as R¹ and wherein R⁸ and R⁹ have the same meanings as R⁵and R⁶ and R⁸ and R⁹ may both be hydrogen.

[0019] In a preferred embodiment, the Linden et al. invention involvesthe administration of a Type IV phosphodiesterase (PDE) inhibitor incombination with the A_(2A) adenosine receptor agonist. The Type IVphosphodiesterase (PDE) inhibitor can be racemic and optically active4-(polyalkoxyphenyl)-2-pyrrolidones of the following formula:

[0020] (disclosed and described in U.S. Pat. No. 4,193,926) wherein R¹⁸and R¹⁹ each are alike or different and are hydrocarbon radicals havingup to 18 carbon atoms with at least one being other than methyl, aheterocyclic ring, or alkyl of 1-5 carbon atoms which is substituted byone or more of halogen atoms, hydroxy, carboxy, alkoxy, alkoxycarbonylor an amino group; amino; R′ is a hydrogen atom, alkyl, aryl or acyl;and X is an oxygen atom or a sulfur atom.

[0021] Rolipram is an example of a suitable Type IV phosphodiesterase orPDE inhibitor included within the above formula. Rolipram has thefollowing structure:

[0022] The present invention is based on the inventors' discovery thatimproved effective treatment of inflammatory disease is achieved by theadministration of certain agonists of A_(2A) adenosine receptors incombination with rolipram or rolipram derivatives that are Type IVphosphodiesterase or PDE inhibitors.

SUMMARY OF THE INVENTION

[0023] Accordingly, one object of the present invention is to provide anovel and improved method for treating inflammatory diseases.

[0024] It is another object of the present invention to provide noveland improved compositions for the treatment of inflammatory disease.

[0025] These and other objects, which will become better understoodduring the course of the following detailed description, have beenachieved by the inventors' discovery of improved compositions andmethods for effectively treating inflammatory diseases by administrationof an agonist of an A_(2A) adenosine receptor in combination withrolipram or a rolipram derivative that is a Type IV phosphodiesterase(PDE) inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] A more complete appreciation of the invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings, wherein:

[0027]FIG. 1 illustrates the relative potencies of adenosine analogs tomodulate TNF_(α)-primed fMLP-stimulated polymorphonuclear cell (PMN)chemiluminescence as a measure of PMN production of oxidative products(0, no TNFα; Δ, WRC-0474[SHA 211]+TNFα; □, CGS 21680+TNFα; and ▴,adenosine+TNFα);

[0028]FIG. 2 illustrates the synergistic effect of WRC-0474[SHA 211] and4-(3cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (rolipram) ininhibiting TNFα-primed (10 U/ml), fMLP-stimulated (100 nM) PMNsuperoxide production: 0, no4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone; ▴, 3 nM4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone; □, 30 nM4-(3-cyclopentyloxy4-methoxyphenyl)-2-pyrrolidone; and, 300 nM4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone.

[0029]FIG. 3 illustrates the synergistic effect of WRC-0474[SHA 211] androlipram in inhibiting TNFα-stimulated adherent PMN superoxide release;

[0030]FIG. 4 illustrates the effect of WRC-0474[SHA 211] and rolipram onTNFα-stimulated PMN adherence to a fibrinogen coated surface;

[0031]FIG. 5 illustrates synergy between A_(2A) adenosine receptoragonists and Rolipram in inhibiting superoxide release fromTNFα-stimulated adherent human neutrophils;

[0032]FIG. 6 illustrates the effects of WRC-0470 and rolipram on theoxidative activity of neutrophils in whole blood;

[0033]FIG. 7 illustrates the effects of WRC-0470 and rolipram on therelease of TNFα from adherent human monocytes and that this activity isdependent on binding of the adenosine agonist to A_(2A) adenosinereceptors.

[0034]FIG. 8 illustrates the effect of WRC-0470 on white blood cellpleocytosis in rats.

[0035]FIG. 9 illustrates the effect of WRC-0470 on blood-brain-barrierpermeability in rats;

[0036]FIG. 10 illustrates the effect of rolipram on white blood cellpleocytosis in rats; and

[0037]FIG. 11 illustrates the combined effect of WRC-0470 and rolipramon white blood cell pleocytosis in rats.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Thus, in a first embodiment, the present invention provides amethod for treating inflammatory diseases by administering an effectiveamount of a compound of formula (I):

[0039] wherein X is a group selected from the group consisting of —OR¹,—NR²R³, and —NH—N═R⁴;

[0040] wherein R¹ is C₁₋₄-alkyl; C₁₋₄-alkyl substituted with one or moreC₁₋₄-alkoxy groups, halogens (-fluorine, chlorine, or bromine), hydroxygroups, amino groups, mono(C₁₋₄-alkyl)amino groups, di(C₁₋₄-alkyl)aminogroups, or C₆₋₁₀-aryl groups (wherein the aryl groups may be substitutedwith one or more halogens (fluorine, chlorine, or bromine), C₁₋₄-alkylgroups, hydroxy groups, amino groups, mono(C₁₋₄-alkyl)amino groups, ordi(C₁₋₄ alkyl)amino groups); C₆₋₁₀-aryl; or C₆₋₁₀-aryl substituted withone or more halogens (fluorine, chlorine, or bromine), hydroxy groups,amino groups, mono(C₁₋₄-alkyl)amino groups, or di(C₁₋₄ alkyl)aminogroups, or C₁₋₄-alkyl groups;

[0041] one of R² and R³ has the same meaning as R¹ and the other ishydrogen;

[0042] R⁴ is a group having the formula (II)

[0043] wherein each of R⁵ and R⁶ independently may be hydrogen,C₃₋₇-cycloalkyl, or any of the meanings of R¹ , provided that R⁵ and R⁶are not both hydrogen;

[0044] Examples of suitable C₆₋₁₀-aryl groups include phenyl andnaphthyl.

[0045] Preferably the compound of formula (I) has X being a group of theformula (III)

—O—CH₂_(n)—Ar  (III)

[0046] wherein n is an integer from 1-4, preferably 2, and Ar is aphenyl group, tolyl group, naphthyl group, xylyl group, or mesitylgroup. Most preferably Ar is a para-tolyl group and n=2.

[0047] Even more preferably, the compound of formula (IV) has X being agroup of the formula (I)

—NH—N═CHCy  (IV)

[0048] wherein Cy is a C₃₋₇-cycloalkyl group, preferably cyclohexyl or aC₁₋₄ alkyl group, preferably isopropyl.

[0049] Specific examples of such compounds of formula (I) includeWRC-0470, WRC-0474 [SHA 211], WRC-0090 and WRC-0018, shown below:

[0050] Of these specific examples, WRC-0474[SHA 211] and WRC-0470 areparticularly preferred.

[0051] Such compounds may be synthesized as described in: Hutchinson, A.J., et al., J. Pharmacol. ExD. Ther., vol. 251, pp. 47-55 (1989);Olsson, R. A., et al., J. Med. Chem., vol. 29, pp. 1683-1689 (1986);Bridges, A. J., et al., J. Med. Chem., vol. 31, pp. 1282-1285 (1988);Hutchinson, A. J., et al., J. Med. Chem., vol. 33, pp. 1919-1924 (1990);Ukena, M., et al., J. Med. Chem., vol. 34, pp. 1334-1339 (1991);Francis, J. E., et al., J. Med. Chem., vol. 34, pp. 2570-2579 (1991);Yoneyama, F., et al., Eur. J. Pharmacol., vol. 213, pp. 199-204 (1992);Peet, N. P., et al., J. Med. Chem., vol. 35, pp. 3263-3269 (1992); andCristalli, G., et al., J. Med. Chem., vol. 35, pp. 2363-2368 (1992); allof which are incorporated herein by reference.

[0052] The present method includes the administration of a Type IVphosphodiesterase (PDE) inhibitor in combination with the compound offormula (I). Examples of Type IV phosphodiesterase inhibitors includethose disclosed in U.S. Pat. No. 4,193,926, and WO 92-079778, andMolnar-Kimber, K. L., et al., J. Immunol., vol. 150, p. 295A (1993), allof which are incorporated herein by reference.

[0053] Specifically, the suitable Type IV phosphodiesterase (PDE)inhibitors include racemic and optically active4-(polyalkoxyphenyl)-2-pyrrolidones of general formula (V)

[0054] (disclosed and described in U.S. Pat. No. 4,193,926) wherein R¹⁸and R¹⁹ each are alike or different and are hydrocarbon radicals havingup to 18 carbon atoms with at least one being other than methyl, aheterocyclic ring, or alkyl of 1-5 carbon atoms which is substituted byone or more of halogen atoms, hydroxy, carboxy, alkoxy, alkoxycarbonylor an amino group or amino.

[0055] Examples of hydrocarbon R¹⁸ and R¹⁹ groups are saturated andunsaturated, straight-chain and branched alkyl of 1-18, preferably 1-5,carbon atoms, cycloalkyl and cycloalkylalkyl, preferably of 3-7 carbonatoms, and aryl and aralkyl, preferably of 6-10 carbon atoms, especiallymonocyclic.

[0056] Examples of alkyl are methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, pentyl, 2-methylbutyl, 2,2-dimethylpropyl, hexyl,heptyl, octyl, nonyl, 1,2-dimethylheptyl, decyl, undecyl, dodecyl andstearyl, with the proviso that when one of R¹⁸ and R¹⁹ is methyl, theother is a value other than methyl. Examples of unsaturated alkyl groupsare alkenyl and alkynyl, e.g., vinyl, 1-propenyl, 2-propenyl, 2-propynyland 3-methyl-2-propenyl.

[0057] Examples of cycloalkyl and cycloalkylalkyl which preferablycontain a total of 3-7 carbon atoms are cyclopropyl, cyclopropylmethyl,cyclopentyl and cyclohexyl.

[0058] Examples of aryl and aralkyl are phenyl and benzyl, which arepreferred, and tolyl, xylyl, naphthyl, phenethyl and 3phenylpropyl.

[0059] Examples of heterocyclic R¹⁸ and R¹⁹ groups are those wherein theheterocyclic ring is saturated with 5 or 6 ring members and has a single0, S or N atom as the hetero atom, e.g., 2- and 3-tetrahydrofuryl, 2 and3-tetrahydropyranyl, 2- and 3-tetrahydrothiophenyl, pyrrolidino, 2- and3-pyrrolidyl, piperidino, 2-, 3- and 4-piperidyl, and the correspondingN-alkyl-pyrrolidyl and piperidyl wherein alkyl is of 1-4 carbon atoms.Equivalents are heterocyclic rings having fewer or more, e.g., 4 and 7,ring members, and one or more additional hetero atoms as ring members,e.g., morpholino, piperazino and N-alkylpiperazino.

[0060] Examples of substituted alkyl R¹⁸ and R¹⁹ groups, preferably of1-5 carbon atoms, are those mono- or polysubstituted, for example, byhalogen, especially fluorine, chlorine and bromine. Specific examples ofsuch halogen-substituted alkyl are 2-chloroethyl, 3-chloropropyl,4-bromobutyl, difluoromethyl, trifluoromethyl,1,1,2-trifluoro-2-chloroethyl, 3,3,3-trifluoropropyl,2,2,3,3,3-pentafluoropropyl and 1,1,1,3,3,3-hexafluoro-2-propyl.Examples of other suitable substituents for such alkyl groups arehydroxy groups, e.g., 2-hydroxyethyl or 3-hydroxypropyl; carboxy groups,e.g., carboxymethyl or carboxyethyl; alkoxy groups, wherein each alkoxygroup contains 1-5 carbon atoms, e.g., ethoxymethyl, isopropoxymethyl,2-methoxyethyl, 2-isopropoxyethyl, 2-butyoxyethyl, 2-isobutyoxyethyl,and 3-pentoxypropyl.

[0061] Also suitable as preferably terminal-positioned substituents onalkyl groups of 1-5 carbon atoms are alkoxycarbonyl of 1-5 carbon atomsin the alkoxy group. Examples of such alkoxycarbonyl substitutedalkyl-groups are ethoxycarbonylmethyl and 2-butoxycarbonylethyl.

[0062] Alkyl groups of 1-5 carbon atoms can also be substituted, e.g.,in the β, Υ and preferably terminal position with amino groups whereinthe nitrogen atom optionally is mono- or disubstituted by alkyl,preferably of 1-5 carbon atoms, or is part of a 4- to 7-membered ring.

[0063] Rolipram and its analogues are specific examples of preferredType IV phosphodiesterase inhibitors.

[0064] Examples of inflammatory diseases which may be treated accordingto the present invention include:

[0065] autoimmune diseases such as lupus erythenatosis, multiplesclerosis, type I diabetes mellits, Crohn's disease, ulcerative colitis,inflammatory bowel disease, osteoporosis, arthritis, allergic diseasessuch as asthma, infectious diseases such as sepsis, septic shock,infectious arthritis, endotoxic shock, gram negative shock, toxicshock,cerebral malaria, bacterial meningitis, adult respiratory distresssyndrome (ARDS), TNFα-enhanced HIV replication and TNFα inhibition ofreverse transciptase inhibitor activity, wasting diseases (cachexiasecondary to cancer and HIV), skin diseases like psoriasis, contactdermatitis, eczema, infectious skin ulcers, cellulitis, organ transplantrejection (including bone marrow, kidney, liver, lung, heart, skinrejection), graft versus host disease, adverse effects from amphotericinB treatment, adverse effects from interleukin-2 treatment, adverseeffects from OKT3 treatment, adverse effects from GM-CSF treatment,adverse effects of cyclosporine treatment and adverse effects ofaminoglycoside treatment, ischemia, mucositis, infertility fromendometriosis, circulatory diseases induced or exacerbated by aninflammatory response such as atherosclerosis, peripheral vasculardisease, restenosis following angioplasty, inflammatory aortic aneurysm,ischemia/reperfusion damage, vasculitis, stroke, congestive heartfailure, hemorrhagic shock, vasospasm following subarachnoid hemorrhage,vasospasm following cerebrovascular accident, pleuritis, pericarditis,and encephalitis.

[0066] The exact dosage of the compound of formula (I) to beadministered will, of course, depend on the size and condition of thepatient being treated, the exact condition being treated, and theidentity of the particular compound of formula (I) being administered.However, a suitable dosage of the compound of formula (I) is 0.5 to 100μg/kg of body weight, preferably 1 to 10 μg/kg of body weight.Typically, the compound of formula (I) will be administered from 1 to 8,preferably 1 to 4, times per day.

[0067] The preferred mode of administration of the compound of formula(I) may also depend on the exact condition being treated. However, mosttypically, the mode of administration will be oral, topical,intravenous, parenteral, subcutaneous, or intramuscular injection.

[0068] Of course, it is to be understood that the compound of formula(I) may be administered in the form of a pharmaceutically acceptablesalt. Examples of such salts include acid addition salts. Preferredpharmaceutically acceptable addition salts include salts of mineralacids, for example, hydrochloric acid, sulfuric acid, nitric acid, andthe like; salts of monobasic carboxylic acids, such as, for example,acetic acid, propionic acid, and the like; salts of dibasic carboxylicacids, such as maleic acid, fumaric acid, oxalic acid, and the like; andsalts of tribasic carboxylic acids, such as, carboxysuccinic acid,citric acid, and the like. In the compounds of formula (I) in which R is—CO₂H, the salt may be derived by replacing the acidic proton of the—CO₂H group with a cation such as Na⁺, K⁺, NH⁺ ₄ mon-, di, tri, ortetra(C₁₋₄-alkyl)ammonium, or mono-, di-, tri-, ortetra(C₂₋₄alkanol)ammonium.

[0069] It is also to be understood that many of the compounds of formula(I) may exist as various isomers, enantiomers, and diastereomers andthat the present invention encompasses the administration of a singleisomer, enantiomer, or diastereomer in addition to the administration ofmixtures of isomers, enantiomers, or diastereomers.

[0070] The compounds of formula (I) can be administered orally, forexample, with an inert diluent with an edible carrier. They can beenclosed in gelatin capsules or compressed into tablets. For the purposeof oral therapeutic administration, the compounds can be incorporatedwith excipients and used in the form of tablets, troches, capsules,elixirs, suspensions, syrups, waters, chewing gums, and the like. Thesepreparations should contain at least 0.5% by weight of the compound offormula (I), but the amount can be varied depending upon the particularform and can conveniently be between 4.0% to about 70% by weight of theunit dosage. The amount of the compound of formula (I) in suchcompositions is such that a suitable dosage will be obtained. Preferredcompositions and preparations according to the present invention areprepared so that an oral dosage unit form contains between about 30 μgand about 5 mg, preferably between 50 to 500 μg, of active compound.

[0071] Tablets, pills, capsules, troches, and the like can contain thefollowing ingredients: a binder, such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient, such as starch or lactose; adisintegrating agent, such as alginic acid, Primogel, corn starch, andthe like; a lubricant, such as magnesium stearate or Sterotes; aglidant, such as colloidal silicon dioxide; a sweetening agent, such assucrose, saccharin or aspartame; or flavoring agent, such as peppermint,methyl salicylate, or orange flavoring. When the dosage unit form is acapsule it can contain, in addition to the compound of formula (I), aliquid carrier, such as a fatty oil.

[0072] Other dosage unit forms can contain other materials that modifythe physical form of the dosage unit, for example, as coatings. Thus,tablets or pills can be coated with sugar, shellac, or other entericcoating agents. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and preservatives, dyes,colorings, and flavors. Materials used in preparing these compositionsshould be pharmaceutically pure and non-toxic in the amounts used.

[0073] For purposes of parenteral therapeutic administration, thecompounds of formula (I) can be incorporated into a solution orsuspension. These preparations should contain at least 0.1% of theaforesaid compound, but may be varied between 0.5% and about 50% of theweight thereof. The amount of active compound in such compositions issuch that a suitable dosage will be obtained. Preferred compositions andpreparations according to the present invention are prepared so that aparenteral dosage unit contains between 30 μg to 5 mg, preferablybetween 50 to 500 μg, of the compound of formula (I).

[0074] Solutions or suspensions of the compounds of formula (I) can alsoinclude the following components: a sterile diluent, such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents: antibacterial agents, suchas benzyl alcohol or methyl parabens; antioxidants, such as ascorbicacid or sodium bisulfite; chelating agents, such asethylenediaminetetraacetic acid; buffers, such as acetates, citrates orphosphates; and agents for the adjustment of tonicity, such as sodiumchloride or dextrose. The parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

[0075] Effective amounts of the Type IV phosphodiesterase inhibitor canbe administered to a subject by any one of various methods, for example,orally as in a capsule or tablets, topically, or parenterally in theform of sterile solutions. The Type IV phosphodiesterase inhibitors,while effective themselves, can be formulated and administered in theform of their pharmaceutically acceptable addition salts for purposes ofstability, convenience of crystallization, increased solubility, and thelike.

[0076] Preferred pharmaceutically acceptable addition salts includesalts of mineral acids, for example, hydrochloric acid, sulfuric acid,nitric acid, and the like; salts of monobasic carboxylic acids, such as,for example, acetic acid, propionic acid, and the like; salts of dibasiccarboxylic acids, such as maleic acid, fumaric acid, oxalic acid, andthe like; and salts of tribasic carboxylic acids, such as,carboxysuccinic acid, citric acid, and the like.

[0077] The Type IV phosphodiesterase may be administered in the form ofa pharmaceutical composition similar to those described above in thecontext of the compound of formula (I).

[0078] While dosage values will vary with the specific disease conditionto be alleviated, good results are achieved when the Type IVphosphodiesterase inhibitor is administered to a subject requiring suchtreatment as an effective oral, parenteral or intravenous dose asdescribed below.

[0079] For oral administration, the amount of active agent per oraldosage unit usually is 0.1-20 mg, preferably 0.5-10 mg. The daily dosageis usually 0.1-50 mg, preferably 1-30 mg. p.o. For parenteralapplication, the amount of active agent per dosage unit is usually0.005-10 mg, preferably 0.01-5 mg. The daily dosage is usually 0.01-20mg, preferably 0.02-5 mg i.v. or i.m.

[0080] With topical administration, dosage levels and their relatedprocedures would be consistent with those known in the art, such asthose dosage levels and procedures described in U.S. Pat. No. 5,565,462to Eitan et al., which is incorporated herein by reference.

[0081] It is to be understood, however, that for any particular subject,specific dosage regimens should be adjusted to the individual need andthe professional judgement of the person administering or supervisingthe administration of the Type IV phosphodiesterase inhibitor. It is tobe further understood that the dosages set forth herein are exemplaryonly and that they do not, to any extent, limit the scope or practice ofthe present invention.

[0082] In a particularly preferred embodiment, the compound of formula(I) and the Type IV phosphodiesterase inhibitor are coadministeredtogether in a single dosage unit. The compound of formula (I) and thetype IV phosphodiesterase inhibitor may be administered in the same typeof pharmaceutical composition as those described above in the context ofthe compound of formula (I).

[0083] By coadministering a Type IV phosphodiesterase inhibitor with theagonist of the A_(2A) adenosine receptor it is possible to dramaticallylower the dosage of the A_(2A) adenosine receptor agonist and the TypeIV phosphodiesterase inhibitor due to a synergistic effect of the twoagents. Thus, in the embodiment involving coadministration of the A_(2A)adenosine receptor agonist with the type IV phosphodiesterase inhibitor,the dosage of the A_(2A) adenosine receptor agonist may be reduced by afactor of 5 to 10 from the dosage used when no type IV phosphodiesteraseinhibitor is administered. This reduces the possibility of side effects.

[0084] The present invention will now be described in more detail in thecontext of the coadministration of WRC-0470, WRC-0474[SHA 211], WRC-0090or WRC-0018 and rolipram. However, it is to be understood that thepresent invention may be practiced with other compounds of formula (I)and other Type IV phosphodiesterase inhibitors of formula (V).

[0085] The present studies establish that anti-inflammatory doses haveno toxic effects in animals; the effect of WRC-0470 to inhibitneutrophil activation is synergistic with rolipram; and intravenousinfusion of WRC-0470 profoundly inhibits extravasation of neutrophils inan animal model of inflammation, an action also synergistic withrolipram. Further, the present studies establish that activation ofA_(2A) receptors on human monocytes strongly inhibits TNFα (aninflamatory cytokine) release. This mechanism further contributes to theanti-inflamatory action of the A_(2A) adenosine receptor agonists of thepresent invention.

[0086] Other features of the invention will become apparent in thecourse of the following descriptions of exemplary embodiments which aregiven for illustration of the invention and are not intended to belimiting thereof.

EXAMPLES

[0087] Materials and Methods

[0088] Materials: f-Met—Leu—Phe(fMLP), luminol, and trypan blue werefrom Sigma Chemical. Ficoll-hypaque was purchased from Flow Laboratories(McLean, A) and Los Alamos Diagnostics (Los Alamos, N. Mex.). Hanksbalanced salt solution (HBSS), and limulus amebocyte lysate assay kitwere from Whittaker Bioproducts (Walkersville, Md.). Human serum albumin(HSA) was from Cutter Biological (Elkhart, Ind.). Recombinant humantumor necrosis factor-alpha was supplied by Dianippon Pharmaceutical Co.Ltd. (Osaka, Japan). ZM241385 was a gidt of Dr. Simon Poucher, ZenecaPharmaceuticals (Chesire, England).

[0089] Leukocyte Preparation: Purified PMN (˜98% PMN and >95% viable bytrypan blue exclusion) containing <1 platelet per 5 PMN and <50 pg/mlendotoxin (limulus amebocyte lysate assay) were obtained from normalheparinized (10 Units/ml) venous blood by a one step ficoll-hypaqueseparation procedure (Ferrante, A., et al., J. Immunol. Meth., vol. 36,p. 109, (1980)). Residual RBC were lysed by hypotonic lysis with iced 3ml 0.22% sodium chloride solution for 45 seconds followed by 0.88 ml of3% sodium chloride solution.

[0090] Chemiluminescence: Luminol enhanced chemiluminescence, a measureof neutrophil oxidative activity, is dependent upon both superoxideproduction and mobilization of the granule enzyme myeloperoxidase. Thelight is emitted from unstable high-energy oxygen species generated byactivated neutrophils. Purified PMN (5×10⁵/ml) were incubated in HBSScontaining 0.1 % human serum albumin (1 ml) with or without adenosine,adenosine analogs, and TNFα (1 U/mL) for 30 minutes at 37° C. in ashaking water bath. Then luminol (1×10⁻⁴M) enhanced f-met-leu-phe (1 μM)stimulated chemiluminescence was read with a Chronolog Photometer(Chrono-log Corp., Havertown, Pa.) at 37° C. for 8 min.Chemiluminescence is reported as relative peak light emitted (=height ofthe curve) compared to samples with TNF and without adenosine oradenosine analogs. WRC-0474[SHA 211] was 10 times more potent thaneither adenosine (ADO) or CGS21680 in decrease TNFα-primedf-met-leu-phe-stimulated PMN chemiluminescence (see FIG. 1).

[0091] Synergy of A_(2A) Adenosine Receptor Agonist andPhosphodiesterase Inhibitors. The synergy between WRC-0474[SHA 211] and4-(3-cyclopentyloxy-4methoxyphenyl)-2-pyrrolidone (rolipram) wasexamined by measuring the effect of combined WRC-0474[SHA 211] androlipram on TNF-primed f-met-leu-phe-stimulated suspended neutrophilsuperoxide release and on the oxidative burst of neutrophils adhering tomatrix proteins (in this model the PMN oxidative burst is enhanced bysmall concentrations of TNFα [e.g. 1 U/ml] when added prior to theaddition of a second stimulus such as the peptide f-met-leu-phe).

[0092] Suspended PMN Superoxide Release: Human PMN (1×10⁶/ml) fromFicoll-Hypaque separation were primed for 30 minutes (37° C.) with orwithout rhTNF (10 U/ml), with adenosine deaminase (1 U/ml), and with orwithout 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone and SHA 211.Cytochrome c (120 μM), catalase (0.062 mg/ml) and fMLP (100 nM) wereadded and the samples incubated for 10 minutes more at 37° C. SOD (200U/ml) was added to matched samples. The samples were iced andcentrifuged (2000 g×10 minutes). The optical density of the supernatantswere read at 550 nm against the matched SOD samples, and the moles ofSOD-inhibitable superoxide released in 10 minutes were calculated.

[0093] A synergistic effect of WRC-0474[SHA 211] and rolipram indecreasing the TNFα-primed fMLP-stimulated PMN oxidative burst wasobserved (see FIG. 2).

[0094] TNFα-stimulated superoxide release of PMN adherent to a matrixprotein (fibrinogen) coated surface: Human PMN (1×10⁶/ml) fromFicoll-Hypaque separation were incubated for 90 minutes in 1 ml of Hanksbalanced salt solution containing 0.1% human serum albumin, cytochrome c(120 μM), and catalase (0.062 mg/ml) in the presence and absence ofrhTNF (1 U/ml), WRC-0474[SHA 211] (10 nM) and rolipram (100 nM) in atissue culture well which had been coated overnight with humanfibrinogen. SOD (200 U/ml) was added to matched samples. Thesupernatants were iced and centrifuged (2000 g×10 minutes) to remove anyremaining suspended cells, and the optical density of the supernatantswere read at 550 mn against the matched SOD samples, and the nmoles ofSOD-inhibitable superoxide released in 90 minutes were calculated.

[0095] A synergistic effect of WRC-0474[SHA 211] and rolipram indecreasing the TNFα-stimulated release of superoxide from PMN adherentto fibrinogen was observed (see FIG. 3).

[0096] Effect of WRC-0474[SHA 211] with and without rolipram onTNF-Stimulated PMN Adherence to a Fibrinogen-Coated Surface. Cronsteinet al., J. Immunol., vol. 148, p. 2201 (1992) reported that adenosinebinding to A₁ receptors increases PMN adherence to endothelium andmatrix proteins and binding to A₂ receptors decreases adherence to thesesurfaces when the PMN are stimulated with fMLP. Despite this, othershave failed to see much of an effect of adenosine (10 μM) onTNFα-stimulated PMN adherence to matrix proteins. In contrast, adenosinedramatically decreases the oxidative burst of TNFα-stimulated PMNadhering to matrix proteins (DeLa Harpe, J., J. Immunol., vol. 143, p.596 (1989)). The experiments described above establish that WRC-0474[SHA211] decreases TNF-stimulated oxidative activity of PMN adhering tofibrinogen, especially when combined with rolipram.

[0097] PMN adherence to fibrinogen was measured as follows as adaptedfrom Hanlon, J. Leukocyte Biol., vol. 50, p. 43 (1991). Twenty-four wellflat-bottomed tissue culture plates were incubated (37° C.) overnightwith 0.5 ml of fibrinogen (5 mg/ml) dissolved in 1.5% NaHCO₃. The plateswere emptied and each well washed 2× with 1 ml of normal saline. Thewells were then filled with 1 ml of HBSS-0. 1% human serum albumincontaining PMN (1×10⁶/ml) with and without rhTNFα(1 U/ml), adenosinedeaminase (ADA) (1 U/ML), WRC-0474[SHA 211] (10 nM), CGS21680 (30 nM),adenosine (100 nM) and rolipram (100 nM). The plates were incubated for90 minutes at 37° C. in 5% CO₂. Following incubation the tissue culturewells were washed free of non-adherent cells with normal saline. Theadherent monolayer of PMN was lysed with 0.1% triton-X, the amount oflactic dehydrogenase (LDH) released from the monolayer assayed (LDH kit,Sigma Co., St. Louis, Mo.), and compared to a standard curve relatingthe LDH content to PMN numbers. The results are shown in FIG. 4.

[0098] As a comparison to WRC-0474[SHA 211] (at only 10 nM), CGS21680(30 nM) decreased TNF-stimulated adherence in the presence of ADA from38% to 30% adhered (p=0.004) (see FIG. 4), and ten times as muchadenosine (100 nM) decreased adherence to 28% adhered (p=0.009 comparedto TNF in the presence of ADA).

[0099] Additional effects of adenosine A_(2A) agonists on adherent humanneutrophil oxidative activity. The bioactivity of test compoundsWRC-0474[SHA 211], WRC-0470, WRC-0090 and WRC-0018 were evaluatedaccording to the following method modified from Sullivan, G. W. et al.,Int. J. Immunonopharmacol, 1995, 17:793-803. Neutrophils (1×10⁶/ml) fromFicoll-Hypaque separation were incubated for 90 minutes in 1 ml of Hanksbalanced salt solution containing 0.1% human serum albumin, cytochrome c(120 μM) and catalase (0.062 mg/ml) in the presence and absence ofrhTNFα (1 U/ml), WRC-0474[SHA 211], WRC-0470, WRC-0090 and WRC-0018(3-300 nM), and rolipram (100 nM) in a tissue culture well which hadbeen coated overnight with human fibrinogen. The supernatants were icedand centrifuged (200 g×10 min) to remove any remaining suspended cells,and the optical densities of the supernatants were read at 550 nmagainst matched superoxide dismutase (SOD) (200 U/ml) samples. Thenmoles of SOD=inhabitable superoxide released in 90 min were calculated.

[0100]FIG. 5 shows synergy between A_(2A) adenosine agonists androlipram in inhibiting TNFα-stimulated adherent PMN oxidative activity(p<0.05). WRC-0474[SHA 211] (30-300 nM), WRC-0470 (300 nM), WRC-0090(300 nM) and WRC-0018 (300 nM) combined with rolipram synergisticallydecreased superoxide release (p<0.05). All four compounds had someactivity in the presence of rolipram. WRC-0474[SHA 211] and WRC-0470were the most active. Nanomolar concentrations of WRC-0474[SHA 211]resulted in biphasic activity. All compounds were synergistic withrolipram to decrease TNFα-stimulated adherent PMN oxidative activity.

[0101] PMN degranulation (adherent cells). The following methods wereadapted from Sullivan, G. W. and G. L. Mandell, Infect. Inumun., 1980:30:272-280. Neutrophils (3.1×10⁶/ml) from Ficoll-Hypaque separation wereincubated for 120 minutes in 1 ml of Hanks balanced salt solutioncontaining 0.1% human serum albumin, ±rh TNFα (10 U/ml), ±WRC-0470(3-300 nM), and ±rolipram (300 nM) in a tissue culture well which hadbeen coated overnight with human fibrinogen. The supernatant fluids withany suspended neutrophils were harvested following incubation,centrifuged (2000×g for 10 min) to remove any suspended cells and thecell-free supernatants frozen. Release of lysozyme, a component ofneutrophil primary and secondary granules was assayed. Lysis of asuspension of Micrococcus lysodeikticus by the “cell-free supernatant”was measured by spectrophotometric analysis (540 mm) to determine theamount of release of granule contents to the surrounding medium.

[0102] Results showed that WRC-0470 (300 nM) with rolipram (300 nM)significantly decreased TNFα-stimulated adherent neutrophildegranulation 67%; P=0.027. The data indicate that in addition todecreasing TNFα-stimulated PMN adherent and the oxidative burst of theseadherent neutrophils, WRC-0470 also decreases degranulation activatedPMN adhering to a biological surface.

[0103] PMN oxidative activity in whole blood. The following methods wereadapted from Rothe, G. A. et al., J. Immunol. Meth. 1991; 138:133-135).Heparinized whole blood (0.8 ml) was incubated (37°; 30 min) withadenosine deaminase (ADA, 1 U/ml), catalase (14,000 U/ml),±dihydrorhodamine 123, ±WRC-0470 (3-300 nM), ±rolipram (300 nM) and±TNFα (10 U/ml). The primed blood samples were stimulated with fMLP (15min), then iced, the red blood cells lysed with FACS lysing solution(Becton-Dickinson, San Jose, Calif.), washed and the leukocytesresuspended in phosphate buffered saline (PBS) These samples containingmixed leukocytes were gated for neutrophils by forward and side scatterand the fluorescence of 10,000 neutrophils measured in the FL1 channelof a FACScan (Beckton-Dickinson) fluorescence activated cell sorter.

[0104] The results are reported as relative mean florescence intensityin FIG. 6 of the drawings. WRC-0470 decreased oxidative activity ofTNFα-primed fMLP-stimulated neutrophils in whole blood and actedsynergistically with rolipram. WRC-0470 (30-300 nM) decreased neutrophiloxidative activity synergistically with rolipram (300 nM) in samplesstimulated with fMLP and in blood samples primed with TNFα and thenstimulated with fMLP.

[0105] Production of TNFα by purified human adherent monocytes. Amonocyte rich monolayer (>95% monocytes) was prepared by incubating 1 mlof the mononuclear leukocyte fraction (5×10⁵/ml) from a Ficoll-Hypaqueseparation in wells of a 24 well tissue culture plate (1 hr; 37° C.; 5%CO₂). The non-adherent leukocytes were removed by washing and culturemedium added to the wells (1 ml RPMI 1640 containing 1.5 mM HEPES-1%autologous serum with penicillin and streptomycin (250 U/ml and 250μg/ml, respectively) and ADA (1 U/ml) ±WRC-0470 (30-100 nM), ±endotoxin(10 ng/ml), +rolipram (300 nM) and ±the adenosine A_(2A) selectiveantagonist4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3a]-[1,3,5]trazinyl-amino]ethyl)-phenol(ZM241385) (50 nM). The samples were incubated for 4 hr (37° C.; 5% CO₂)and the supernatants harvested. Any suspended cells were removed bycentrifugation and the cell-free samples frozen (−70° C.). TNFα wasassayed in the cell-free supernatants by an ELISA kit (CistronBiotechnology, Pine Brook, N.J.).

[0106] As shown in FIGS. 7A and 7B, WRC-0470 ±rolipram-decreasedendotoxin-stimulated adherent monocyte production of TNFα (P<0.050). Asillustrated in FIG. 7B, the A_(2A) selective antagonist ZM241385significantly inhibited the effect of WRC-0470 (300 nM) combined withrolipram (300 nM) (p=0.020) on TNFα release from monocytes. Hence,WRC-0470 affects TNFα-stimulated neutrophil activity and decreasesendotoxin-stimulated TNFα production by monocytes.

[0107] Effects of WRC-0470 and rolipram on the extravasation of whiteblood cells in a rat model of inflammation. Adult wistar rats(approximately 200 g) were anesthetized with intermuscular injections ofketamine and xylazine. Bacteria meningitis (BM) was induced viaintracisternal inoculation of either E. coli strain 026:B6LPS (200 ng),cytokines (IL-1 and TNFα, or LPS plus cytokines). The animals were theninfused with rolipram and/or WRC-1470 over the duration of theexperiment using a Harvard pump. CSF (cerebrospinal fluid) and blood wasthen sampled at 4 h postinoculation and alterations in BBBP (blood-brainbarrier permeability) and WBC (white blood cell) counts were determined.CSF and WBC concentrations were determined with standard hemacytometermethods. For assessment of % BBBP, rats were given an intravenousinjection of 5 μCi 125I-labeled bovine serum albumin concomitant withintracisternal inoculation. Equal samples of CSF and blood were readsimultaneously in a gamma counter and after subtraction of backgroundradioactivity, % BBBP was calculated by the following formula: %BBBP=(cpm CSF/cpm blood)×100. All statistical tests were performed usingInstat biostatistical software to compare the post-inoculation samplesof experimental rats with the control rats. The statistical tests usedto generate p-values were Student's t-test and ANOVA.

[0108] Results of the tests are reported in FIGS. 8 and 9. Infusion ofWRC-0470 at a rate of 0.005-1.2 μg/kg/hr inhibited pleiocytosis (p<0.05as compared to control). The effect of WRC-0470 on BBBP is shown in FIG.9. A significant response is seen with a range of 0.01-0.015 μg/kg/hr(p<0.05 as compared to control). A rebound effect is noted with theadministration of 1.2 μg/kg/hr where % BBBP returned to control. FIG. 10shows the effect of rolipram on CSF pleocytosis in a range of 0-0.01μg/kg/hr with 0.01 μg/kg/hr inhibiting 99% of the pleocytosis (p<0.05).Rolipram at either 0.01 or 0.005 μg/kg/hr showed significant inhibitionof alterations of BBBP (p<0.05), while a dose of 0.002 μg/kg/hr had nosignificant effect

[0109] The effect of a combination of rolipram and WRC-0470 on CSF WBCpleocytosis is illustrated in FIG. 11. Rolipram (0.001 μg/kg/hr) incombination with WRC-0470 (0.1 μg/kg/hr) inhibited migration of WBC's(200±70 WBC/μl) into the sub-arachnoid space (SAS) to a greater extentthan did either rolipram (1,670±1,273 WBC/μl, p<0.050) or WRC-0470(600±308 WBCs/μl, p<0.050) alone. The data show a powerful inhibitingeffect of WRC-0470 and a synergy with rolipram to prevent inflammationin an animal model.

[0110] Application of A_(2A) adenosine receptors with or withoutrolipram on balloon angioplasty and gene therapy. Balloon angioplasty iscommonly used to treat coronary artery stenosis. Restenosis followingballoon angioplasty (BA) occurs in up to 40% of coronary interventions.Holmes et al., American Journal of Cardiology, 53: 77C-81C (1984).(40%). Restenosis results from a complex interaction of biologicprocesses, including (i) formation of platelet-rich thrombus; (ii)release of vasoactive and mitogenic factors causing migration andproliferation of smooth muscle cells (SMC); (iii) macrophage and otherinflammatory cell accumulation and foam cell (FC) formation; (iv)production of extracellular matrix; and (v) geometric remodeling.Recently the use of coronary stents and pharmacologic intervention usinga chimeric antibody to block the integrin on platelets have beenpartially successful in limiting restenosis after percutaneous coronaryinterventions in man. Topol et al., Lencet, 343: 881-886 (1994). Sinceinflammatory cell infiltration might be central to the response toinjury, and restenotic processes, and adenosine, activing via A_(2A)adenosine receptors, inhibits tissues inflammatory cell accumulation, wehypothesize that agonists of A_(2A) adenosine receptors±type IV PDEinhibitors will reduce the incidence of restenosis following balloonangioplasty.

[0111] In addition, recent advances in local delivery catheters and genedelivery techniques raise the interesting and exciting possibility ofadministering genes locally into the vessel wall. Nabel et al., Science,249: 1285-1288 (1990); Leclerc et al., Journal of ClinicalInvestigation, 90: 936-944 (1992). Adenoviral-mediated gene transferaffords several advantages over other techniques. However, geneexpression is only transient, and has been observed for 7-14 days withdiminuition or loss of expression by 28 days. Lack of persistence mayresult from host immune cytolytic responses directed against infectedcells. The inflammatory response generated by the present generation ofadenovirus results in neoitimal lesion formation and may thus offset thebenefit of a therapeutic gene. Newman et al., Journal of ClinicalInvestigation, 96: 2955-2965 (1995). An A_(2A) adenosine receptoragonist±a type IV phosphodiesterase inhibitor in combination withadenovirus may improve the efficiency of gene transfer.

[0112] Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that, within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed:
 1. A method of treating inflammatory disease,comprising administering to a patient in need thereof an agonist of anA_(2A) adenosine receptor in combination with rolipram, a rolipramderivative or other compound that is a Type IV phosphodiesteraseinhibitor.
 2. The method of claim 1, wherein said disease is selectedfrom the group consisting of: autoimmune diseases (lupus erythenatosis),multiple sclerosis, type I diabetes mellits, Crohn's disease, ulcerativecolitis, inflammatory bowel disease, osteoporosis, arthritis, allergicdiseases (asthma), infectious diseases (sepsis),septic shock, infectiousarthritis, endotoxic shock, gram negative shock, toxic shock,cerebralmalaria, bacterial meningitis, adult respiratory distress syndrome,TNFα-enhanced HIV replication and TNFα inhibition of reversetransciptase inhibitor activity, wasting diseases (cachexia secondary tocancer and HIV), skin diseases (psoriasis), contact dermatitis, eczema,infectious skin ulcers, cellulitis,organ transplant rejection, graftversus host disease, adverse effects from amphotericin B treatment,adverse effects from interleukin-2 treatment, adverse effects from OKT3treatment, adverse effects from GM-CSF treatment, adverse effects ofcyclosporine treatment and adverse effects of aminoglycoside treatment,ischemia, mucositis, infertility from endometriosis, atherosclerosis,peripheral vascular disease, restenosis following angioplasty,inflammatory aortic aneurysm, ischemia/reperfusion damage, vasculitis,stroke, congestive heart failure, hemorrhagic shock, vasospasm followingsubarachnoid hemorrhage, vasospasm following cerebrovascular accident,pleauritis, pericarditis, and encephalitis.
 3. The method of claim 1,wherein said agonist of an A_(2A) adenosine receptor has the formula (I)

wherein X is a group selected from the group consisting of —OR¹, —NR²R³,and —NH—N═R⁴; wherein R¹ is C₁₋₄-alkyl; C₁₋₄-alkyl substituted with oneor more C₁₋₄-alkoxy groups, halogens (fluorine, chlorine, or bromine),hydroxy groups, amino groups, mono(C₁₋₄-alkyl)amino groups,di(C₁₋₄-alkyl)amino groups, or C₆₋₁₀-aryl groups (wherein the arylgroups may be substituted with one or more halogens (fluorine, chlorine,or bromine), C₁₋₄-alkyl groups, hydroxy groups, amino groups,mono(C₁₋₄-alkyl)amino groups, or di(C₁₋₄alkyl)amino groups); C₆₋₁₀-aryl;or C₆₋₁₀-aryl substituted with one or more halogens (fluorine, chlorine,or bromine), hydroxy groups, amino groups, mono(C₁₋₄-alkyl)amino groups,or di(C₁₋₄ alkyl)amino groups, or C₁₋₄-alkyl groups; one of R² and R³has the same meaning as R¹ and the other is hydrogen; R⁴ is a grouphaving the formula

wherein each of R⁵ and R⁶ independently may be hydrogen,C₃₋₇-cycloalkyl, or any of the meanings of R¹, provided that R⁵ and R⁶are not both hydrogen; or a pharmaceutically acceptable salt thereof. 4.The method of claim 1, wherein said agonist of an A_(2A) adenosinereceptor is selected from the group consisting of:


5. The method of claim 1, wherein said Type IV phosphodiesteraseinhibitor is a compound having formula (V):

wherein R¹⁸ and R¹⁹ each are alike or different and are hydrocarbonradicals having up to 18 carbon atoms with at least one being other thanmethyl, a heterocyclic ring, or alkyl of 1-5 carbon atoms which issubstituted by one or more of halogen atoms, hydroxy, carboxy, alkoxy,alkoxycarbonyl or an amino group; or amino.
 6. The method of claim 1,wherein said type IV phosphodiesterase inhibitor is rolipram.
 7. Themethod of claim 1, wherein said agonist of an A_(2A) adenosine receptoris

and said Type IV phosphosterase inhibitor is rolipram.
 8. The method ofclaim 1, wherein said agonist of an A_(2A) adenosine receptor is


9. The method of claim 1, wherein said A_(2A) adenosine receptor agonistand said Type IV phosphosterase inhibitor are coadministered together tothe patient in need thereof.
 10. A pharmaceutical composition comprisingan effective amount of an agonist of an A_(2A) adenosine receptor incombination with rolipram or a rolipram derivative or a rolipramanalogue or other Type IV phosphodiesterase inhibitors.
 11. Thepharmaceutical composition of claim 10, wherein said agonist of anA_(2A) adenosine receptor has the formula (I)

wherein X is a group selected from the group consisting of —OR¹, —NR²R³,and —NH—N═R⁴; wherein R¹ is C₁₋₄-alkyl; C₁₋₄-alkyl substituted with oneor more C₁₋₄-alkoxy groups, halogens (fluorine, chlorine, or bromine),hydroxy groups, amino groups, mono(C₁₋₄-alkyl)amino groups,di(C₁₋₄-alkyl)amino groups, or C₆₋₁₀-aryl groups (wherein the arylgroups may be substituted with one or more halogens (fluorine, chlorine,or bromine), C₁₋₄-alkyl groups, hydroxy groups, amino groups,mono(C₁₋₄-alkyl)amino groups, or di(C₁₋₄alkyl)amino groups); C₆₋₁₀-aryl;or C₆₋₁₀-aryl substituted with one or more halogens (fluorine, chlorine,or bromine), hydroxy groups, amino groups, mono(C₁₋₄-alkyl)amino groups,or di(C₁₋₄ alkyl)amino groups, or C₁₋₄-alkyl groups; one of R² and R³has the same meaning as R¹ and the other is hydrogen; R⁴ is a grouphaving the formula

wherein each of R⁵ and R⁶ independently may be hydrogen,C₃₋₇-cycloalkyl, or any of the meanings of R¹, provided that R⁵ and R⁶are not both hydrogen; or a pharmaceutically acceptable salt thereof.12. The pharmaceutical composition of claim 10, wherein said agonist ofan A_(2A) adenosine receptor is selected from the group consisting of


13. The pharmaceutical composition of claim 10, wherein said Type IVphosphodiesterase inhibitor is a compound having formula (V):

wherein R¹⁸ and R¹⁹ each are alike or different and are hydrocarbonradicals having up to 18 carbon atoms with at least one being other thanmethyl, a heterocyclic ring, or alkyl of 1-5 carbon atoms which issubstituted by one or more of halogen atoms, hydroxy, carboxy, alkoxy,alkoxycarbonyl or an amino group; amino; R′ is a hydrogen atom, alkyl,aryl or acyl; and X is an oxygen atom or a sulfur atom.
 14. Thepharmaceutical composition of claim 10, wherein said type IVphosphodiesterase inhibitor is rolipram.
 15. The pharmaceuticalcomposition of claim 10, wherein said agonist of an A_(2A) adenosinereceptor is

and said Type IV phosphosterase inhibitor is rolipram.
 16. Thepharmaceutical composition of claim 10, wherein said agonist of anA_(2A) adenosine receptor is

and said Type IV phosphosterase inhibitor is rolipram.
 17. The method ofclaim 1, further comprising administering said agonist of an A_(2A)adenosine receptor in combination with rolipram, a rolipram derivativeor other compound that is a Type IV phosphodiesterase inhibitor duringand for a limited time after balloon angioplasty to reduce frequence andextent of restenosis.
 18. The method of claim 1, further comprisingadministering said agonist of an A_(2A) adenosine receptor incombination with rolipram, a rolipram derivative or other compound thatis a Type IV phosphodiesterase inhibitor in conjunction with a genedelivery modality to limit inflammation and thereby improve efficiencyand stability of gene therapy.
 19. The method of claim 1, wherein saidgene delivery modality is selected from the group comprising viruses andlipid vesicles.